Hemolytic Component Research Articles

Complement in chronic secretory otitis media. C3 breakdown and C3 splitting activity.

Occurrence of in vivo C3 breakdown and in vitro C3 splitting activity was studied in serum and middle-ear effusion (MEE) samples from 30 children with chronic secretory otitis media (SOM). The MEE showed strongly elevated levels of both low- and high-molecular-weight C3 breakdown products, along with decreased factor B, C4, and C3 levels. Total hemolytic complement component activity was virtually absent from MEE. The MEE fluids were found to contain C3 splitting factors as demonstrated by their high capacity to convert C3 in vitro from fresh normal human serum. This activity was not inhibited by the classic complement pathway inhibitor, 0.01M ethylene glycol tetra-acetic acid with 0.005M magnesium chloride. The results suggest that a strong local complement activation has taken place and that the factors responsible are present in the MEE of patients with SOM.

Immunopathological Studies of Polyarteritis Nodosa and Wegener's Granulomatosis: A Report of 43 Patients with 51 Renal Biopsies

Although it is generally considered that vasculitis of the polyarteritis nodosa (PAN) group and Wegener's granulomatosis (WG) is immune complex (IC) mediated, there are no simultaneous data on circulating IC, complement levels and deposits of Ig and complement in the kidney. Therefore we have performed a retrospective study of 43 patients suffering from PAN and WG. Ig glomerular deposits were uncommon and scanty, except in two patients with WG; C3 deposits were detected in 12 patients, whereas fibrinogen was constantly found when lesions were recent and active. Similar data were obtained for the renal vessel walls. Contrasting with these results, rheumatoid factors and cryoglobulins, suggestive of the presence of circulating IC, were detected respectively in nine of 39 and seven of 37 patients, and IC 'activity' assessed by the Raji cell assay and the Clq binding assay was found respectively in six of 17 and nine of 10 patients before treatment, and in none of 10 and five of seven patients in remission. Haemolytic complement activity and complement components were never decreased, but the C3d breakdown product of C3 was elevated in all the eight patients studied before treatment. Signs of persistent hepatitis B virus (HBV) infection were detected in five of 25 patients of the PAN group, whereas three of eight patients with WG had only anti-HBV antibodies. Furthermore, cytomegalovirus (CMV) could be isolated from the blood in a case of WG before the treatment was started. Persistent interferonaemia was detected in one of five patients. These results suggest either that renal deposition of CIC is transient, the paucity of Ig deposits being due to rapid clearance of IC by phagocytic cells; or alternatively that vascular and glomerular lesions are not caused by CIC, as in some cases of experimental vasculitis induced by infectious agents.

Latency and heterogeneity of Entamoeba histolytica hemolysins.

We have found that the hemolytic potency of P30 - a subcellular fraction that contains most of the hemolytic activity of Entamoeba histolytica trophozoites--increased up to 100 times by preincubation for 36 h at 36 degrees C. This remarkable increase in potency was due to the in vitro generation of heat-labile as well as heat-stable hemolytic components and could be prevented if P30 was preincubated at 4 degrees C or subjected to either heating at 90 degrees C or to repeated freeze-thawing cycles before preincubation at 36 degrees C. The major hemolytic activity of E. histolytica thus appears to depend on latent, heat-labile and heat-stable hemolysins that are probably unmasked through the action of endogenous amebal enzymes.

Characteristics of a hemolysin from Gonyaulax monilata Howell

A toxic extract from the red tide dinoflagellate, Gonyaulax monilata Howell has been isolated. The hemolytic activity of the extract against human erythrocytes was compared, in vitro, to those of digitonin and phospholipase A 2. The dose-response curves of the toxic extract and digitonin were statistically parallel to one another; however, digitonin exhibited a potency four times that of the extract on a weight basis. The potency of the phospholipase was much lower than either the digitonin or the toxic extract and the slope of the dose-response curve differed considerably from those of the latter two compounds. No erythrocyte ghosts were present following hemolysis with any of the compounds utilized. The hemolytic component of the toxic extract appears to cause fragmentation of the erythrocyte membrane. It does not appear that hemolysis is caused by hydrolytic action on the lecithin fraction of the membrane. The active component exhibits chemical characteristics like those of an unsaturated glycolipid, possibly steroid based.

Effects of the toxin of red tide, Ptychodiscus brevis, on canine tracheal smooth muscle: a possible new asthma-triggering mechanism

The red tide toxin produced by Ptychodiscus brevis becomes airborne by the thrashing action of the surf and wind and induces cough, rhinorrhea, watery eyes, and sneezing in normal humans and wheezing in asthmatic patients. The mechanism of the contractile response induced by P. brevis toxin (PBTX) was investigated with isolated canine tracheal smooth muscle. Tetrodotoxin and atropine blocked the contractile effect of PBTX, and neostigmine potentiated the contraction. Mepyramine, phentolamine, methysergide, and chlorisondamine did not inhibit the effect of PBTX. This is the first description of a naturally occurring airborne substance that causes smooth muscle contraction by stimulating the axon sodium channels, resulting in the release of acetylcholine at postganglionic parasympathetic efferent nerve endings. The in vitro effect of PBTX on canine tracheal smooth muscle indicates that PBTX is capable of causing respiratory irritation and thus may precipitate an asthmatic attack. It is possible, however, that the mechanism in vivo may also include stimulation of a cough receptor reflex and/or stimulation of sodium channels of afferent vagus nerve fibers. In vitro evidence suggests that isoproterenol, atropine, and verapamil may be used to eliminate or prevent the respiratory symptoms that follow exposure to airborne red tide toxin. The use of high-pressure liquid chromatography separated fractions indicates that the neurotoxic component, not the hemolytic component, is responsible for contractions.

Hemolytic assay for venom phospholipase A 2

A rapid and sensitive spectrophotometric assay for venom phospholipase A 2 based on the hemolysis of guinea pig erythrocytes in the presence of decomplemented serum and cardiotoxin (direct lytic factor) is described. This assay is particularly useful for rapid multisample analyses, such as those used in monitoring chromatography fractions, and is specific for phospholipase A 2 in she presence of other potentially hemolytic venom components. The hemolytic mechanism is shown to be a combination of the action of lysophospholipids liberated from lipoproteins in the serum and the synergistic action of phospholipase A 2 and cardiotoxin on the erythrocyte membrane.

XX/XY Lymphoid Chimerism in a Boy With Fatal Lymphohistiocytic Proliferation

XX/XY chimerism, present in peripheral blood and bone marrow cells, but absent in other somatic tissues, was demonstrated in a 6-year-old patient. The initial clinical presentation with "butterfly" rash, pancytopenia with a hemolytic component, and hypergammaglobulinemia was suggestive of an immune disorder. Infiltration of skin, bone marrow, and lymph nodes with histiocytes, without disruption of the general configuration, was similar to the findings in familial lymphohistiocytosis. The results of specific cellular markers and of cytogenetic and immunologic studies are analyzed and the nature of the disease and origin of the chimeric state are discussed. The most likely explanations include a chronic graft-vs-host reaction induced by proliferation of foreign lymphoid cells derived from a blood transfusion that the child had received during infancy, or proliferation of neoplastic cells present in the transfused blood.

A hemolytic principle associated with the red-tide dinoflagellate Gonyaulax monilata

A hemolytic component has been found associated with cells from unialgal cultures of the red-tide dinoflagellate Gonyaulax monilata. Gel filtration chromatography increased the specific hemolytic activity of the active principle 26-fold. As well as being a potent hemolysin, the active component is lethal to cockroaches, guppies and white mice.

Possible involvement of red cell membrane proteins in the hemolytic action of Portuguese Man-of-War toxin

Glycophorin and the fragments isolated from trypsinizing intact rat, dog, sheep and human red blood cells (rbc's) neutralize the hemolytic action of the Portuguese Man-of-War venom. This action can be blocked by rabbit antisheep hemolysin and phytohemagglutinin, a lectin which preferentially binds to glycophorin. Concanavalin A, which binds to band-3 protein of rbc membranes, does not block the neutralizing action of rbc tryptic fragments or glycophorin. The concentrations of rat, dog, human and sheep glycophorin which half neutralize venom induced hemolysis are inversely and linearly proportional to the hemolytic sensitivities of these rbc's to the venom. These data implicate glycophorin as a possible binding site for the hemolytic component of the Portuguese Man-of-War venom.

Host-tumor interactions: basis for the anemia in rats bearing the hepatoma 7777.

A significant anemia develops in Buffalo rats bearing the Morris hepatoma 7777. By indiceal measurements the red blood cells (RBCs) appear to be microcytic and hypochromic. Ferrokinetic studies demonstrate a decreased uptake of iron in the bone marrow and an increased incorporation of iron in the spleens of the tumor-bearing animals. RBC survival studies indicate the presence of a hemolytic component which appears to contribute to the anemia. The RBCs from tumor-bearing animals (T-RBC) have a greatly increased fragility in sodium chloride solutions. Inspection of these T-RBCs by electron microscopy demonstrates significant echinocytosis when they are compared to normal cells. Electrophoretic separation of the hemoglobin from T-RBCs shows a pattern different from normal cells and consistent with the pattern described for hemoglobin from spleen and bone marrow. The findings are consistent with bone marrow inadequacy and an increased splenic erythropoiesis which is insufficient to maintain normal hematological values. The hypothesis is presented that the RBCs synthesized under these circumstances appear in the circulation with an "immature" hemoglobin pattern and are hemolyzed more readily. This process then contributes to the hemolytic component and development of the anemia.

Quantitation of serum complement components and plasma C3d in patients with malignant lymphoma: relation to the stage of the tumor and circulating immune complexes.

Total hemolytic complement activity (CH 50) and complement component levels were measured in 27 patients with Hodgkin's disease and 31 patients with non-Hodgkin malignant lymphoma. CH 50 values were higher than normal in almost all the patients. Increased levels of serum C4, C3 and factor B were observed in 62%, 31% and 19% of these patients, respectively. However, plasma concentration of C3d, a breakdown product of C3, was elevated in 29% of the cases. The hypercatabolism of C3 was not closely associated with the presence of circulating immune complexes, as assessed by the C1q binding assay, nor with the presence of general symptoms in the patients. On the contrary, it appeared to be in relation with the extent of the malignant disease.

Search for relationships among the hemolytic, phospholipolytic, and neurotoxic activities of snake venoms

Several snake venom neurotoxins are larger and more complex than the well-studied group of postsynaptic toxins exemplified by alpha-bungarotoxin. Several of these, exemplified by beta-bungarotoxin, show phospholipase A2 activity (phosphatide 2-acylhydrolase, EC 3.1.1.4) when tested in the presence of detergents. The high hemolytic activity of crotoxin, the neurotoxin of Crotalus durissus terrificus, in the presence of lecithin has been attributed to this activity. The phospholipase A2 activity of several snake venom proteins has now been compared under the physiological conditions of the hemolysis tests. It appears that only the basic component of crotoxin, B, is enzymatically active, and that its activity is not inhibited by component A under these conditions, or in the presence of deoxycholate. Phosphatidylserine is found to be digested more readily than egg white phosphatidylcholine; and also causes hemolysis in conjunction with much lower levels of crotoxin. In neither case is calcium required or stimulating. Phospholipase from Crotalus adamanteus, which is not neurotoxic, digests phosphatidylcholine more rapidly than does crotoxin, but phosphatidylserine more slowly; yet it is slightly less active than crotoxin in the hemolysis test with phosphatidylcholine, and much less with phosphatidylserine. The digestion of several phospholipids by either enzyme fails to release the expected protons in the absence of detergents at 37 degrees .beta-Bungarotoxin, highly neurotoxic, has negligible phospholipase A2 activity in the absence of detergents, and is almost nonhemolytic in conjunction with all phospholipids tested.Binding studies with (125)I-labeled compounds show that rabbit erythrocytes and ghosts have much greater affinity for crotoxin than for beta-bungarotoxin and do not bind Crotalus adamanteus phospholipase. The crotoxin complex is split in the course of binding, with only component B, the hemolytic component, becoming bound. It appears that the role of component A may be to diminish the nonspecific binding tendency of component B. Our data appear to be consistent with the concepts that affinity to membranes, particularly to specific sites on synaptic membranes, is the critical requirement for beta type neurotoxicity, and that this property, at least in some instances, has evolved from phospholipase A2 enzymes, but does not necessarily require retention and expression of enzymatic activity.

Alternative pathway of complement in sickle cell disease.

Thirty-one patients, 10 months to 20 years of age, were studied. A complement abnormality was not identified in sera from patients with sickle cell disease (SCD) by the methods employed in the present study. Concentrations of C3, factor B, total hemolytic activity (CH50), properdin, and C3b inactivator were similar in sera from patients and control subjects (Table 1 and Fig. 2). Although concentrations of C3b inactivator protein were below normal in a few patients, there was no evidence that these levels were low enough to alter the functions mediated by this protein. Initiation of the complement sequence via the alternative pathway by reaction with inulin was equal in patient and control sera when assessed by the activation of factor B, cleavage of C3 and the comsumption of hemolytic complement components (Table 1). Lysis of erythrocytes treated with reduced glutathione was similar in patient and control sera during alternative pathway activation (Fig. 3), indicating comparable formation of lytic complexes via this pathway. An abnormality of the alternative pathway was not detected when the serum from patients with sickle cell disease was reacted with inulin. Thus, this polysaccharide, although commonly empolyed to assess alternative pathway function, is not satisfactory for studying serum from these patients. In addition, activation of the alternative pathway by cobra venom factor was comparable with controls when assessed by the lysis of glutathione-treated erythrocytes.

Hereditary spherocytosis and glucose-6-phosphate dehydrogenase deficiency

The first reported case of hereditary spherocytosis (HS) and glucose-6-phosphate dehydrogenase deficiency in a black is presented. The recent literature is reviewed, with emphasis on the frequency of multiple inherited RBC defects in this ethnic group. Despite a coexisting hemoglobinopathy or enzyme deficiency, HS can be diagnosed in most cases by the peripheral blood smear, osmotic fragility curve, and family history. The implications of the double RBC abnormality are discussed, stressing the importance of splenectomy in relieving the hemolytic component due to spherocytosis.

Hereditary Spherocytosis and Glucose-6-Phosphate Dehydrogenase Deficiency

The first reported case of hereditary spherocytosis (HS) and glucose-6-phosphate dehydrogenase deficiency in a black is presented. The recent literature is reviewed, with emphasis on the frequency of multiple inherited RBC defects in this ethnic group. Despite a coexisting hemoglobinopathy or enzyme deficiency, HS can be diagnosed in most cases by the peripheral blood smear, osmotic fragility curve, and family history. The implications of the double RBC abnormality are discussed, stressing the importance of splenectomy in relieving the hemolytic component due to spherocytosis.

Leucocyte function in a case of chronic benign neutropenia of infancy associated with circulating leucoagglutinins.

A case of chronic benign neutropenia is described in association with circulating leucoagglutinins. IgG and IgM leucoagglutinins demonstrated by Sephadex G-200 chromatography agglutinated neutrophils and monocyte enriched leucocyte preparations. Total IgG, IgA, IgM and IgE, total haemolytic complement, C3 and C4 complement components were all within normal limits. Lymphocyte populations and transformation were normal apart from a relative increase in the number of B lymphocytes. In vitro studies showed that the patient's monocytes responded in chemotaxis, phagocytosis and intracellular killing of S. aureus in a comparable fashion to that of control neutrophils. Although the patient's serum also agglutinated monocytes these findings do not exclude the possibility that leucoagglutinins may have aetiological significance in this disease. The study also emphasizes the versatility of the monocyte in benign neutropenia.

Multiple forms of staphylococcal alpha-toxin.

A group of proteins was readily extracted at neutrality from trichloroacetic acid precipitates of staphylococcal culture filtrate supernatants, while alpha-toxin was dissolved and activated by treating the precipitate with 8 M urea, with acidic buffers or by heating to 90-100 degrees C at neutrality. Heat activation of the precipitate produced a relatively pure alpha-toxin with a molecular weight of 39,000. alpha-Toxin was eluted together with three other proteins on hydroxyl apatite chromatography, and evidence was obtained for an association between the four proteins. On isoelectric focusing a haemolytic fraction was obtained at pH 6.2, probably due to acid activation of the precipitate formed at the cathodic end of the column. The alpha-haemolytic fractions with pI's of 7.4 and 8.6 were shown to consist of alpha-toxin only when analyzed by acrylamide electrophoresis in the presence of sodium dodecyl sulphate. The haemolytic component with a pI of 9.2 contained two additional components of molecular weights of 27,500 and 18,000. Chromatography of this material on Sephadex G-200 showed that alpha-toxin and the two proteins appeared as a high molecular complex.

The Identification and Purification of Multiple Forms of o-Haemolysin (o-Toxin) of Clostridium perfringens Type A

The theta-haemolysin of Clostridium perfringens was purified from culture supernatant fluids of type A strains by fractional ammonium sulphate precipitation and isoelectric focusing in narrow pH 5 to 8 gradients. Four components detected on electrofocusing were designated theta-1(pI6-8to6-9),theta-2(pI6-5to6-6),theta-3(pI6-1to6-3) and theta-4(pI5-7to5-9). Specific activities ranged from 0-4 x 10-6 to 1-2 x 10-6 haemolytic units/mg protein and 2950 to 3600 LD-50/mg protein. Each haemolytic component was activated by cysteine hydrochloride, and inactivated by cholesterol, by addition of sheep erythrocyte ghosts and by heating at 60 degrees C for 10 min; mouse erythrocytes were more resistant than sheep erythrocytes to haemolysis. A reaction of identity was obtained between components in gel diffusion. Sodium dodecyl sulphate polyacrylamide discgel electrophoresis gave molecular weights in the range 59,000 to 62,000 for each component. A similar value was obtained for theta-1 on density gradient ultracentrifugation. Although the multiple forms were free of 11 factors present in culture supernatants, crossed immunoelectrophoresis and disc gel electrophoresis revealed minor contaminants. These studies reveal that theta-haemolysin has physical properties in common with other oxygen-labile haemolysins.

慢性肝疾患における溶血補体価および補体成分に関する研究

慢性肝疾患における溶血補体価および補体成分に関する研究

Erythrocyte deformability in uremic hemodialyzed patients.

Erythrocyte deformability was measured in normal people and in patients with chronic renal failure treated with hemodialysis. This was accomplished by the measurement of flow rate through polycarbonate filters with pore diameters of 3 microns. Erythrocytes with altered flow rates were found in five of seven splenectomized patients with chronic renal failure. Splenectomized individuals with normal renal function did not show this effect on the erythrocytes. The cells of 14 other patients on dialysis, who had spleens, showed normal flow-rate characteristics. This suggests that part of the hemolytic component of the anemia of chronic renal failure may be associated with cellular alterations that compromise the passage of erythrocytes through the microcirculation of the spleen. The improvement of the hemolytic anemia of uremia after splenectomy may be associated with the persistence of these rigid cells in the circulation.